Novel Junctional and Nuclear Roles of the Mitochondrial Protein Mitofusin 2 During Inflammation

Background Severe inflammation can cause widespread damage to the endothelium and result in fatal illnesses such as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Vascular endothelial barrier dysfunction is the major vascular complication in ALI. One of the key mechanisms of barrier disruption is the disassembly of adherens junction (AJ) complexes consisting of VE‐cadherin as well as accessory proteins such as β‐catenin and p120‐catenin. We observed that Mfn2, which has previously been primarily studied as a mitochondrial protein, is also located at the endothelial plasma membrane and co‐localizes with VE‐cadherin as well as translocates into nucleus under inflammatory condition, suggesting novel roles in AJs. Methods and Results By using three‐dimensional structured illumination microscopy (3D‐SIM) which provides high spatial resolution (□130 nm), we studied Mfn2 localization in human lung microvascular ECs (HLMVECs). Mfn2 predominantly co‐localizes with the mitochondrial membrane protein Tom20 but it also co‐localizes at the plasma membrane with VE‐cadherin in resting ECs. However, upon pro‐inflammatory stimulation with TNF‐α, Mfn2 is dissociated from AJs and no longer co‐localizes with VE‐cadherin. Mechanistically, Mfn2 directly interacts with VE‐cadherin and β‐catenin in resting ECs as assessed by immunoprecipitation assay and a proximity ligation assay (PLA). To determine whether Mfn2 affects endothelial barrier integrity and function, Mfn2 was depleted in HLMVECs with a doxycycline inducible lentiviral shMfn2 RNA. Mfn2 silencing disrupted AJ junctions as visualized by VE‐cadherin and β‐catenin immunostaining. Mfn2 depletion significantly increased transendothelial permeability of ECs as assessed by a FITC‐conjugated albumin tracer assay and transendothelial resistance (TER). We also identified novel binding partners of Mfn2 using the high affinity tandem mass spectrometry (LC‐MS/MS). The analysis of gene ontology indicates that Mfn2 interacts with cell‐cell interaction proteins including cadherin binding and nuclear factor binding proteins with >1.5‐fold increase. Finally, the complex of Mfn2 and β‐catenin translocates into the nucleus upon TNF‐α stimulation. Conclusion These data suggest that Mfn2 may directly regulate AJ stability in ECs by interacting with the AJ complex proteins VE‐cadherin and β‐catenin. Mfn2 may act as a co‐regulator with β‐catenin, a key transcription factor in inflammatory condition. These studies enhance our understanding of how the “mitochondrial” protein Mfn2 regulates inflammatory activation via novel “non‐mitochondrial” functions. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .